Taking place shortly after the events of the comic mini-series, Halo: Uprising, Halo 3 begins with the Master Chief entering Earth's atmosphere and crashing to the ground in eastern Africa, where he is found by Sgt. Major Johnson and the Arbiter. The Chief, Johnson, and company fight their way out of the jungle and arrive at a UNSC outpost. Here, Commander Keyes and Lord Hood plan a last-ditch effort to stop the Covenant leader, the High Prophet of Truth from activating a Forerunner artifact uncovered outside the ruins of the city of New Mombasa. The Chief is ordered to clear a way into the city of Voi and to destroy all anti-air Covenant defenses so Hood can lead the last of Earth's ships against the Prophet. Using the opening caused by the ground attack, Hood mounts an offensive against Truth's ship, but the Prophet activates the buried artifact which creates an enormous slipspace portal. As the human ships recover from the shock wave, Truth and his followers enter the portal, while a ship controlled by the Flood crash-lands nearby. Elite forces, allied with humanity, arrive and vitrify Flood-infected areas of Earth, neutralizing the parasitic threat. Following the cryptic message from the human artificial intelligence Cortana left aboard the Flood cruiser, the Chief, Arbiter, Elites, Johnson, Keyes and a handful of Marines follow Truth through the portal. Joining them is the Forerunner construct 343 Guilty Spark, who aids the Chief as he no longer has any function to fulfill after the destruction of his Halo installation in Halo: Combat Evolved. Traveling through the portal, the humans and Elites discover an immense artificial structure, the Ark far beyond the edges of the Milky Way galaxy. Here Truth can activate all the Halos. The Chief and company activate the installation's map room to find Truth at the Ark's control room. During their journey, the Flood arrive en masse on the former Covenant Holy City High Charity, and begin infesting the installation. While attempting to gain access to The Ark' Control Room, Johnson is captured by Truth; the prophet needs a human to utilize the Forerunner technology. Attempting to rescue Johnson, Keyes is killed by the prophet, and Johnson is forced to activate the rings. The Flood leader Gravemind forges a temporary truce with the Chief and Arbiter in an effort to stop Truth. The Arbiter, Master Chief, and Flood forces arrive and overwhelm Truth's guards, rescuing Johnson and halting the installations' firing. After the Arbiter kills Truth, Gravemind turns on the Chief and Arbiter, who escape the Flood's grasp.
The Chief, Arbiter and Guilty Spark discover that the Ark is creating a new ringworld to replace the one previously destroyed. The Chief decides to activate only this new ring to eliminate the local Flood while sparing the galaxy at large. Before he can activate the ring, however, he needs an Activation Index. Knowing that Cortana acquired a copy of one on the first Halo, he rescues the AI from High Charity and creates a chain reaction to destroy the infested city and severely damage the Gravemind.
Arriving on the new Halo, Cortana warns that the Gravemind is trying to rebuild itself on the ring. The Chief, the Arbiter, and Johnson make their way to the control room, where they will activate Halo's weapon. Guilty Spark explains that because the ring is not yet complete, a premature activation will destroy it and the Ark. When Johnson ignores his warning, Guilty Spark kills him to protect "his" ring. The Chief destroys Guilty Spark, activates the ring, and escapes the ring's blast on a frigate, Forward Unto Dawn.
Only the front half of Forward Unto Dawn, carrying the Arbiter, makes it through the portal. Believing the Chief and Cortana to have perished, a memorial service is held for the fallen heroes of the human and Covenant war. After the memorial service, the Arbiter departs for his home planet, where the Elites are finally free of the Prophets' hegemony. Meanwhile, the rear half of Forward Unto Dawn floats in unknown space. Cortana drops a beacon, but realizes it may be years before they are rescued. The Chief enters cryonic sleep, telling Cortana to "wake me, when you need me." If the game is completed at the Legendary difficulty level, the scene continues to show the piece of Forward Unto Dawn drifting towards a mysterious planet.